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PDBsum entry 1guj
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Insulin at ph 2: structural analysis of the conditions promoting insulin fibre formation.
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Authors
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J.L.Whittingham,
D.J.Scott,
K.Chance,
A.Wilson,
J.Finch,
J.Brange,
G.Guy dodson.
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Ref.
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J Mol Biol, 2002,
318,
479-490.
[DOI no: ]
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PubMed id
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Abstract
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When insulin solutions are subjected to acid, heat and agitation, the normal
pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the
molecule undergoes conformational changes allowing it to follow an alternative
aggregation pathway (via a monomeric species) leading to the formation of
insoluble amyloid fibres. To investigate the effect of acid pH on the
conformation and aggregation state of the protein, the crystal structure of
human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure
reveals that the native fold is maintained at low pH, and that the molecule is
still capable of forming dimers similar to those found in hexameric insulin
structures at higher pH. Sulphate ions are incorporated into the molecule and
the crystal lattice where they neutralise positive charges on the protein,
stabilising its structure and facilitating crystallisation. The sulphate
interactions are associated with local deformations in the protein, which may
indicate that the structure is more plastic at low pH. Transmission electron
microscopy analysis of insulin fibres reveals that the appearance of the fibres
is greatly influenced by the type of acid employed. Sulphuric acid produces
distinctive highly bunched, truncated fibres, suggesting that the sulphate ions
have a sophisticated role to play in fibre formation, rather as they do in the
crystal structure. Analytical ultracentrifugation studies show that in the
absence of heating, insulin is predominantly dimeric in mineral acids, whereas
in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect
of acid on the aggregation state of insulin is also complex. These results
suggest that acid conditions increase the susceptibility of the molecule to
conformational change and dissociation, and enhance the rate of fibrillation by
providing a charged environment in which the attractive forces between the
protein molecules is increased.
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Figure 2.
Figure 2. Negative staining transmission electron
micrographs of human insulin fibres prepared under a variety of
conditions. Unless otherwise stated, all solutions contained 5
mg/ml human insulin. The conditions used were: (a) 0.004 M
H[2]SO[4] (pH 2.1), 70 °C; (b) 0.01 M HCl (pH 2.0), 70
°C; (c) H[3]PO[4] (pH 2.0), 90 °C; (d) 0.5 M citric acid
(pH 1.9), 90 °C; (e) 20 mg/ml bovine insulin in 8.3 M acetic
acid (pH 1.6), 37 °C; (f) 20% acetic acid (pH 2.0) and 0.004
M Na[2]SO[4], 90 °C.
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Figure 3.
Figure 3. Stereo view illustrations showing comparisons of
the pH 2 insulin dimer (green) with the dimer of (a) the T[6]
insulin hexamer (blue),[20.] and (b) the B9 Ser->Glu mutant
insulin dimer (red). [9.] Only C^a atoms and the sulphate ions
(ball and stick representation in the low pH structure) are
shown. The dimers were overlapped using an alignment on residues
B9-B19 and D9-D19, which constitute the B chain a-helices in
each dimer. This Figure was made using MOLSCRIPT.[36.]
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2002,
318,
479-490)
copyright 2002.
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Secondary reference #1
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Title
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Structure of an insulin dimer in an orthorhombic crystal: the structure analysis of a human insulin mutant (b9 ser-->Glu).
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Authors
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Z.P.Yao,
Z.H.Zeng,
H.M.Li,
Y.Zhang,
Y.M.Feng,
D.C.Wang.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 1999,
55,
1524-1532.
[DOI no: ]
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PubMed id
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Figure 7.
Figure 7 Hydrogen-bond network among side chains of B9, B10 and
B13 from both monomers at the dimer-forming surface. Distances
are in Å.
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Figure 8.
Figure 8 Conformational comparison of B-chain N-terminal
residues, showing the O state (a) in the crystal and (b) in
solution. (a) Overlapped structures of B9E HI (red), DPI (blue),
DHPI (green). (b) The 25 NMR structures (green) of engineered
(B1, B10, B16, B27)Glu, des B30-insulin overlapped on B9E HI
(red). The coordinates of DPI, DHPI and (B1, B10, B16, B27)Glu,
des B30-insulin are taken from the Protein Data Bank, with codes
1pid, 1dei and 1hui, respectively.
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The above figures are
reproduced from the cited reference
with permission from the IUCr
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